AbstractAn isolated lung ventilated with pulses of negative pressure and perfused through the pulmonary vasculature was utilized for the study of 3‐sn‐phosphatidylcholine synthesis. The perfusion fluid consisted of a Krebs‐Ringer phosphate buffer with 6% bovine serum albumin, pH 7.4, and the appropriate substrate. The simultaneous incorporation of (1‐14C) palmitate and (2‐3H) glycerol and the simultaneous incorporation of (CH3‐14C) choline and (CH3‐3H) methionine were examined. From these experiments it is concluded: 1) lung tissue incorporates (2‐3H) glycerol into 3‐sn‐phosphatidylcholine to a greater extent than any other lipid examined; 2) both choline and methionine contribute to the synthesis of 3‐sn‐phosphatidylcholine, and 50–70% of the label in its nitrogen base is derived from choline and 30–50% from methionine; and 3) a high PO2 appears to reduce the synthesis of 3‐sn‐phosphatidylcholine.